The HUH Catalytic site

Mechanism and Overall Protein Architecture.

Historically, bacteriophage fX174 protein A (gpA) was the first identified HUH superfamily member (see (Kornberg & Baker, 1992)) although, surprisingly, no structural information is available.

Many related proteins subsequently identified by bioinformatics (Ilyina & Koonin, 1992, Koonin & Ilyina, 1993) included proteins involved in catalysis of viral and plasmid rolling circle replication (RCR), conjugative plasmid transfer, and DNA transposition (Kapitonov & Jurka, 2001, Garcillan-Barcia, et al., 2002, Ronning, et al., 2005, Ton-Hoang, et al., 2005, Toleman, et al., 2006, Garcillan-Barcia, et al., 2009). They all carry conserved protein motifs, including the "HUH" motif composed of two histidine (H) residues separated by a bulky hydrophobic (U) residue, and the Y-motif containing either one or two tyrosine (Tyr) residues (found in Y1 and Y2 enzymes respectively).

Y1 HUH enzymes (Fig 1.41.1) include Rep proteins of some plasmids with ssDNA replication intermediates (such as pUB110 (Gruss & Ehrlich, 1989), a wide range of eukaryotic viruses (Rosario, et al., 2012), most conjugative plasmid relaxases (de la Cruz, et al., 2010, Guglielmini, et al., 2011), ISCR (insertion sequences related to IS91(Toleman, et al., 2006)) and IS200/IS605 insertion sequence family transposases (Ronning, et al., 2005, Ton-Hoang, et al., 2005). Y2 enzymes include fX174 gpA itself, Rep proteins of other isometric ssDNA and dsDNA phages (e.g. phage P2 (Odegrip & Haggard-Ljungquist, 2001)), some cyanobacterial and archaeal plasmids and parvoviruses (e.g. adeno-associated virus, AAV) as well as transposases of the IS91 and helitron families (Kapitonov & Jurka, 2001), and MOB_F family plasmid relaxases. In some cases, both Y residues are mechanistically important while for others, only one of the pair appears to be essential.

HUH enzymes use a unique mechanism for catalysing ssDNA breakage and joining. The active site tyrosine creates a 5'-phosphotyrosine intermediate and a free 3'-OH at the cleavage site (Fig 1.41.1). The 3'-OH can be used for different tasks. The most obvious is to prime replication, as observed for HUH domains in single-stranded phage Rep proteins, RCR plasmids and conjugative relaxases. The 3'-OH group can also act as the nucleophile for strand transfer to resolve the phosphotyrosine intermediate in the termination step of RCR replication, conjugative transfer and transposition.

The HUH enzyme cleavage polarity is inverse to that of the tyrosine recombinases, which make 3' phosphotyrosine intermediates and generate free 5'-OH groups that cannot be used as replication primers (Grindley, et al., 2006). HUH enzymes also require a divalent metal ion to facilitate cleavage by localizing and polarising the scissile phosphodiester bond in contrast to the cofactor-independent tyrosine recombinases. Depending on the enzyme, Mg²⁺, Mn²⁺ or other divalent metal ions can be used in vitro (Datta, et al., 2003, Larkin, et al., 2005, Boer, et al., 2006, Boer, et al., 2009, Hickman, et al., 2010, Edwards, et al., 2013). It is presumed that Mg²⁺ or Mn²⁺ are the physiological cofactors. The HUH histidine pair provides two of the three ligands necessary for metal ion coordination (Fig 1.41.1). The location and identity of the third, invariably polar (Glu, Asp, His or Gln) residue varies across the superfamily.

3D structures of several Rep and relaxase HUH domains with and without bound DNA are available e.g. (Datta, et al., 2003, Guasch, et al., 2003, Larkin, et al., 2003, Hickman, et al., 2004, Boer, et al., 2006, Boer, et al., 2009, Messing, et al., 2012). The order of HUH and Y motifs varies in the primary sequence: in the Relaxase group, the Y-motif is upstream of the HUH-motif whereas in the Rep group it is downstream (Fig1.41.1). This "circular permutation" (Koonin & Ilyina, 1993, Dyda & Hickman, 2003, Guasch, et al., 2003) changes the domain topology. Nevertheless, the three-dimensional constellation of active site residues is virtually identical across the superfamily.

Given the diverse HUH protein functions, it is not surprising that other domains are often appended to the HUH domain (Fig1.41.1). These are often of unknown function but, ATP dependent helicase, zinc binding, primase and multimerisation domains are recurring themes (Petit, et al., 1998, Bruand & Ehrlich, 2000, Odegrip, et al., 2000, Kapitonov & Jurka, 2001, Chang, et al., 2002, Hickman, et al., 2004, Clerot & Bernardi, 2006). For example, the ssDNA substrates needed by HUH enzymes can be generated by a dedicated DNA helicase domain C-terminal to the HUH domain (Im & Muzyczka, 1990, Brister & Muzyczka, 1999, Kapitonov & Jurka, 2001, Clerot & Bernardi, 2006) or alternatively by recruitment of a host helicase (Petit, et al., 1998, Bruand & Ehrlich, 2000, Odegrip, et al., 2000, Chang, et al., 2002). RCR processes use 3'-5'helicase activity acting on the template strand to facilitate DNA unwinding at the replication fork while in conjugation, helicases (as part of the relaxase) are transported into the recipient cell and track 5' to 3' on the transported ssDNA.

DNA Recognition

Many HUH nucleases recognize and bind DNA hairpin structures with cleavage sites located within the hairpin or in the ssDNA on the 5' or 3' side of the stem. The crucial role of hairpins has been firmly established in many systems including plasmid conjugation, eukaryotic viral and plasmid replication and transposition (Orozco & Hanley-Bowdoin, 1996, Brister & Muzyczka, 2000, Ronning, et al., 2005, Ton-Hoang, et al., 2005, Boer, et al., 2006, Messing, et al., 2012, Ton-Hoang, et al., 2012). In other systems, palindromic sequences that can form DNA hairpins are present near the probable HUH nuclease cleavage sites [Feschotte, 2001] (del Solar, et al., 1998). Such hairpins can be formed in vivo under a number of physiological conditions (see (Bikard, et al., 2011)).

Structural studies revealed that small DNA hairpins can be recognized in several different ways: sequence-specific recognition of the dsDNA stem; structure-specific recognition of irregularities in the stem; or sequence-specific recognition of the hairpin loop (Guasch, et al., 2003, Hickman, et al., 2004, Ronning, et al., 2005, Hickman, et al., 2010, Messing, et al., 2012, Edwards, et al., 2013).

The hairpin-flanking DNA - in many cases in single-stranded form - is also often important for recognition. Relaxases, for example, make extensive contacts with the bases extending between the hairpin and cleavage site (Guasch, et al., 2003, Larkin, et al., 2003, Edwards, et al., 2013), and for a relative of IS200/IS605 transposases, TnpA_REP, nucleotides on the 5' side of the hairpin are crucial for binding and sequence-specific recognition (Messing, et al., 2012). Other family members (Hickman, et al., 2004, Ruiz-Maso, et al., 2007), have more complex binding modes.

HUH enzymes as transposases

Transposases of members of the IS200/IS605(Ton-Hoang, et al., 2005), IS91 (Mendiola, et al., 1994) and ISCR (Toleman, et al., 2006) insertion sequence families and the eukaryotic helitrons (Kapitonov & Jurka, 2001) are also HUH enzymes. Those IS200/IS605 family are the best understood.

IS200/IS605 family

IS200/IS605 family transposases are single domain proteins with only the essential HUH motif and a single catalytic Tyr (Y1 transposases, Fig 1.41.1). Both TnpA_IS608 and TnpA_ISDra2 are obligatory dimers and the active sites are believed to adopt two functionally important conformations, one in which each is composed of the HUH motif from one monomer and the Tyr residue carried by an alpha-helix (aD) from the other (trans configuration), and the other in which both motifs are contributed by the same monomer (cis configuration). Only the former has been observed crystallographically.

Similar proteins are sometimes found associated with repeated Extragenic Palindromes (REP sequences whose hairpin structures resemble the ends of IS200/IS605 family members.

RCR transposons: IS91, ISCR and Helitron families

The earliest identified HUH domain transposases were those of the IS91 family (Garcillan-Barcia, et al., 2002) and are significantly larger than Y1 transposases (Fig 1.41.1), carry a Y2 motif and include an N-terminal zinc binding motif and additional domains of yet unidentified function.

A group of related elements, the ISCRs often associated with a variety of antibiotic resistance genes (see (Toleman, et al., 2006)) carry an orf (the CR or common region) resembling IS91 family transposases but with only a single Tyr (Fig 1.41.1). In addition, eukaryotic relatives, the Helitrons, have been identified by bioinformatic approaches.

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